*Purpose: Regulatory T cell (Treg) migration is important for tolerance, although the molecules that regulate Treg entry to lymphatics are poorly understood. Lymphatic endothelial cells (LEC) express high levels of programmed death-ligand 1 (PD-L1), which interacts with PD-1 and CD80 and is involved in tolerance. The interaction of effector T cell CD80, but not PD-1, with LEC PD-L1 is crucial for effector T cell tolerance. Yet how Treg interact with LEC via PD-L1 is not known. Here we investigated the hypothesis that Treg PD-1 signals to LEC PD-L1 to regulate lymphatic transendothelial migration (TEM).

*Methods: Human and murine dermal LECs were used in biochemical, phenotypic, and functional analyses of PD-L1 signaling. Human and murine naïve, activated, and regulatory CD4 T cells were isolated and migrated across LEC in vitro and lymphatic vessels in vivo. Recombinant PD-1 or CD80 extracellular domain fused with IgG1 (PD-1-Fc or CD80-Fc) was used to induce PD-L1 signaling. Signaling was blocked with specific anti-PD-1 or anti-PD-L1 Abs.

*Results: Both human and mouse LEC highly express PD-L1, but not PD-1, on the cell surface. Crosslinking PD-L1 on LEC with PD-1-Fc induced classical NFκB (p65), extracellular-signal-regulated kinase (ERK), and Akt (Thr308) activation. Ligation of PD-L1 on LEC by PD-1-Fc, but not CD80-Fc, decreased the intercellular junction protein VE-cadherin, which accompanies and is important for T cell TEM. The decreased VE-cadherin was recovered by blocking LEC NFκB-NIK, ERK, and PI3K but not NFκB-p65 signaling. LEC PD-L1 ligation also enhanced VCAM-1 expression which was inhibited by blocking NFκB-p65, but not NFκB-NIK, ERK, PI3K/Akt signaling. Similar changes to LEC junctions were observed when Treg, which highly express PD-1, migrated across LEC layers, but non-Treg did not cause the same effect. Blockade of LEC PD-L1 with anti-PD-L1 mAb, but not anti-CD80, inhibited Treg TEM and prevented the decrease in VE-cadherin expression. Pretreatment of mice with anti-PD-L1 mAb inhibited Treg, but not non-Treg, migration into local draining lymph nodes.

*Conclusions: LEC PD-L1 signals through NFκB-NIK, ERKs, and PI3K/Akt (Thr308) to modulate VE-cadherin, and through classical NFκB p65 to regulate VCAM-1 expression. The modulated LEC structures uniquely affect Treg TEM. The data demonstrate an important and novel role for Treg PD-1 and LEC PD-L1 in regulation of lymphatic migration and ultimately Treg suppressive function.

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